Equivalence of the microscopic and macroscopic models of chromatography: stochastic-dispersive versus lumped kinetic model

The microscopic model of chromatography is a stochastic model that consists of two fundamental processes: (i) the random migration of the molecules in the mobile phase, and (ii) the random adsorption-desorption of molecules on the stationary phase contained in a chromatographic column. The diffusion and drift of the molecules in the mobile phase is described with a simple one-dimensional random walk. The adsorption-desorption process is modeled by a Poisson process that assumes exponential sojourn times of the molecules in both the mobile and the stationary phases. The microscopic, or molecular model of chromatography studied here turns out to be identical to the macroscopic lumped kinetic model of chromatography, whose solution is well known in chromatography. A complete equivalence of the two models is established via the identical expressions they provide for the band profiles.     

Hydrophobicity of ionisable compounds studied by countercurrent chromatography

Countercurrent chromatography (CCC) is a liquid chromatography technique in which the stationary phase is also a liquid. The main chemical process involved in solute separation is partitioning between the two immiscible liquid phases: the mobile phase and the support-free liquid stationary phase. The octanol-water partition coefficients (P(o/w)) is the accepted parameter measuring the hydrophobicity of molecules. It is considered to estimate active principle partitioning over a biomembrane. It was related to the substance biological activity. CCC is able to work with an octanol stationary phase and an aqueous mobile phase. In this configuration, CCC is a useful and easy alternative to measure directly the P(o/w) of the molecules compared to other methods including the classical and tedious shake-flask method. Three ketones are used as model compounds to illustrate the CCC protocol of P(o/w) measurement. The focus of this work is put on ionisable molecules whose apparent P(o/w) is completely changed by ionization. β-Blockers, diuretics and sulfonamides are compound classes that were studied. Some of the experimentally determined P(o/w) coefficients of the molecular forms disagreed with calculated and experimental values available in the literature. The P(o/w) coefficients of the ionic forms and the acidity constants were also calculated using a theoretical model. Relationships between biological properties and hydrophobicity are also discussed.     

A STOICHIOMETRIC DISPLACEMENT MODEL FOR RETENTION OF SOLUTE IN LIQUID CHROMATOGRAPHY WITH MULTI-COMPONENT SYSTEM

Considering all the kinds of interactions between solute and solvent, solute and stationary phase, solvent and stationary phase molecules as well as the competitional adsorption among various kinds of solvent molecules on the stationary phase, we present a stoichiometric displacement model of solute retention with four sets of parameters in liquid chromatography. This model was tested with data from both literature and experiments done by ourselves. These results show that this model may fit the experimental data for a liquid chromatography system with various kinds of mobile phases consisting of a complete range of multi-components and with different types of stationary phases.     

Thermodynamic description of retention mechanism in liquid chromatography

The chemical and physical properties of the stationary and the mobile phase determine the retention and selectivity of the separated molecules in chromatographic process. It should explain the specific and non-specific interactions in the chromatographic system. Special emphasis should be placed on column selection through the adequate choice of column dimensions (diameter and length) and the type of stationary phase with required parameters, which contributes the system effectiveness. However, column effectiveness is unable to guarantee a satisfactory level of substance separation. For this reason, the interactions between the analyte, the stationary and the mobile phase need to be investigated to determine the retention factors of the separated substances and the optimal parameters of the chromatographic system. According to the principles of thermodynamics, in particular molecular interactions, the chromatographic process has to be carried out with a high level of control. This work can be treated a brief tutorial devoted to thermodynamics of liquid chromatography process with a special emphasis on molecular interactions between analyte species and the components of the mobile and stationary phases.     

非理想状态下的塔板理论模型

 在过程中 ,将流动相看成是由许多连续的塔板组成 ,每一塔板的高度与固定相塔板的高度相同。初始浓度的溶质被认为全部集中在流动相的第一塔板中 ;溶质在流动相和固定相之间动态分布。由于动力学因素的影响 ,当流动相流过一个塔板距离时 ,溶质不能够迅速地从固定相释放到流动相中 ,因此溶质在流动相和固定相中的分布浓度受到两个因素即反映系统热力学性质的分配系数P和反映系统动力学性质的释放概率因子α的影响。这一过程被认为是非理想状态下的过程。     

Study of dead volume measurement in packed subcritical fluid chromatography with ODS columns and carbon dioxide-modifier mobile phases

Studies were done for providing a simple, rapid and reliable procedure of void volume measurement in packed subcritical fluid chromatography (pSubFC), with CO2-modifier mobile phases containing high modifier amounts. Methods used in RPLC with ODS columns were applied in pSubFC: gravimetric, homologous series linearisation and unretained marker injection. Results lead us to propose the method of marker injection to determine the void volume in pSubFC. Acetonitrile was chosen as the void volume marker among six tested markers. Furthermore, void volume variations vs. the modifier volume (from 5 to 45%) were studied for nine organic modifiers. The void volume variations were related both to adsorption-desorption phenomena between the mobile phase and the stationary phase and to mobile phase density changes. These variations allowed the classification of the modifiers into four groups on the basis of the molecular interactions.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Direct chiral high performance liquid chromatography separation of O-ethyl O-phenylN - isopropylphosphoroamidothioates

The enantiomers of thirty-nine O-ethyl O-phenyl N-isopropyl phosphoroamidothioates have been separated by high performance liquid chromatography on a Pirkle model chiral stationary phase using ten mobile phase. Chromatographic data are presented for the separation of these organic phosphorus enantiomers on the chiral phase. The influences of molecular structures and compositions of mobile phases have been described.     

Supercritical fluid chromatographic solvatochromic modifier polarity studies

To understand the chromatographic process as a whole, whether it be for gas chromatography (GC), liquid chromatography (LC), or supercritical fluid chromatography (SFC), one needs to know the chemical and physical nature of the mobile and stationary phases and also the interactions that take place between analytes (solutes) and the two phases. An approach towards Investigating the ways that stationary and mobile phases contribute to chromatographic retention Involves exploring the effects of solvent polarity on the strength of the mobile phase. In SFC this could involve determining the polarity of several different modifier/carbon dioxide mobile phases. In this paper, the use of a solvatochromic indicator to learn more about the effects of SFC modifier/mobile phase polarity will be investigated and discussed using several different modifiers and a diolmodified silica column.     

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F13C9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C8) and octadecyl (C18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C18 phase, F13C9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C8, C18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C18 or C8 phases and was even disadvantageous on F13C9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.     

Retention properties of acetone-water mobile phases on a biphenylsiloxane-bonded silica stationary phase in reversed-phase liquid chromatography

The solvation parameter model system constants and retention factors were used to interpret retention properties of 39 calibration compounds on a biphenylsiloxane-bonded stationary phase (Kinetex biphenyl) for acetone-water binary mobile phase systems containing 30–70% v/v. Variation in system constants, phase ratios, and retention factors of acetone-water binary mobile phases systems were compared with more commonly used acetonitrile and methanol mobile phase systems. Retention properties of acetone mobile phases on a Kinetex biphenyl column were more similar to that of acetonitrile than methanol mobile phases except with respect to selectivity equivalency. Importantly, selectivity differences arising between acetone and acetonitrile systems (the lower hydrogen-bond basicity of acetone-water mobile phases and differences in hydrogen-bond acidity, cavity formation and dispersion interactions) could be exploited in reversed-phase liquid chromatography method development on a Kinetex biphenyl stationary phase.     

Retention of C60 and C70 fullerenes on reversed-phase high-performance liquid chromatographic stationary phases.

The separation of C60 and C70 fullerenes on four different polysiloxane stationary phases was examined. It was determined that polar solvents can be used as mobile phases effectively for the separation of fullerene molecules. Unlike previously published work, a polymeric octadecyl siloxane (ODS) stationary phase provided higher separation factors for C70/C60 than did monomeric ODS stationary phases or phenyl substituted stationary phases. For example, for a methanol-diethyl ether (50:50, v/v) mobile phase and C60, k' approximately 5.0 separation factors, alpha = 3.3, were achieved with polymeric ODS compared to alpha = 2.2, with a monomeric ODS stationary phase. A linear solvation energy relationship (LSER) was used to model the importance of solvent interactions and stationary phase interaction to solute retention.     

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.     

Modelling of elution-extrusion counter-current chromatography using perfect replacement approach

Basing on the perfect replacement approach the equilibrium cell model is developed to describe the separation process in elution-extrusion counter-current chromatography (EECCC). As is known, EECCC consists of three steps: classical elution, sweeping elution, and extrusion. The perfect replacement approach means that during sweeping elution step, the mobile phase contained in the column moves and interacts with the "old" stationary phase in the same mode as during the classical elution step; the "new" and "old" stationary phases do not mix; and after the contacting with the mobile phase the concentration of solutes in the "old" stationary phase remains constant and this stationary phase volume is pushed ahead to the exit of the column. Equations are presented allowing the simulation of the chromatogram of solutes eluted from the column with the mobile phase during the elution period and the chromatogram of solutes pushed out of the column with the stationary phase during the extrusion period of EECCC. These equations can help to choose the optimal conditions for conducting elution-extrusion counter-current chromatography.     

Dual‐mode hydrophilic interaction normal phase and reversed phase liquid chromatography of polar compounds on a single column

Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the liquid chromatography, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: (i) the complete solid phase moiety; (ii) the solid porous part carrying the active interaction centers; (iii) the volume of the inner column pores. The selective uptake of water from the bulk aqueous‐organic mobile phase significantly affects the volume and the properties of polar stationary phases. Some polar stationary phases provide dual‐mode retention mechanism in aqueous‐organic mobile phases, reversed‐phase in the water‐rich range, and normal‐phase at high concentrations of the organic solvent in water. The linear solvation energy relationship model characterizes the structural contributions of the non‐selective and selective polar interactions both in the water‐rich and organic solvent‐rich mobile phases. The inner‐pore convention provides a single hold‐up volume value for the retention prediction on the dual‐mode columns over the full mobile phase range. Using the dual‐mode monolithic polymethacrylate zwitterionic micro‐columns alternatively in each mode in the first dimension of two‐dimensional liquid chromatography, in combination with a short reversed‐phase column in the second dimension, provides enhanced sample information.     

High-performance liquid chromatography of nucleic acid constituents: Chromatographic examination of novel stationary phases

Summary Silica-bonded stationary phases were developed for the separation of nucleic acid constituents and their properties investigated with homologous oligoriboadenylic acids in electrostatic interaction chromatography and with alkylbenzenes in reversed-phase chromatography. Analysis of retention data confirmed the stratified molecular structure of the surface which consist of a layer of propyl chains anchoredvia siloxane bridges to the silica surface proper and of polar moieties attached to the hydrocarbonaceous functions. The polar top layer contains weak cationic and/or hydrophobic binding sites, is strongly hydrated in contact with aqueous eluents and bars the access by large biopolymers to the hydrocarbonaceous sublayer. In reversed-phase chromatography of small non polar molecules with hydro-organic eluents, however, this layer is accessible and engenders a retentive behavior typical for weak hydro-carbonaceous bonded phases. As a result the stationary phases, depending on the nature of the sample and the mobile phase, exhibit the properties of "soft" phases for the chromatography of biopolymers under mild elution conditions and those of "hard" phases for the separation of small non-polar molecules under conditions generally employed in reversed-phase chromatography. The retention of nucleic acid constituents on most of the stationary phases investigated subject to a dual mechanism as a result of the interplay of electrostatic and hydrophobic interactions between the eluites and the binding sites on the stationary phase surface. Siliceous stationary phases having surface morphology described above are suitable for the separation of nucleic acid constituents having widely ranging molecular weights up to 3 × 10⁶ Daltons provided the support has appropriate pore dimensions. This is demonstrated by the separation of mixtures arising from digesting t-RNA^pha or polyadenylic acids as well as those of ribosomal RNA’s and different forms of the plasmid pBR322 DNA. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

HPLC of synthetic polymers characterization of polystyrenes by high performance precipitation liquid chromatography (HPPLC)

Summary The advantages and disadvantages of high performance precipitation liquid chromatography have been demonstrated for polystyrene homopolymers. Depending on the mobile phase composition at the dissolution point of the polymeric sample and surface properties of the stationary phase, elution is governed either by a solution process or by adsorption. A contribution by adsorption was noticed on silica as well as on reversed phases based on silica with a normal phase gradient of increasing polarity (heptane to dichloromethane). Elution was solely governed by solubility of the polymers on both types of stationary phase for polystyrenes with a molecular weight above 35 000 and reversed phase gradient of decreasing polarity (methanol to dichloromethane). Under these conditions an identical dependence of elution solvent composition on sample size was found as for turbidity titrations. Due to differences in the velocity of the eluent front and the polymeric sample with porous stationary phases the polymers can be eluted as colloidal solutions Non-porous stationary phases are superior in this respect because the velocities of eluent and solutes are identical.     

Retention mechanisms in super/subcritical fluid chromatography on packed columns

Whereas the retention rules of achiral compounds are well defined in high-performance liquid chromatography, on the basis of the nature of the stationary phase, some difficulties appear in super/subcritical fluid chromatography on packed columns. This is mainly due to the supposed effect of volatility on retention behaviours in supercritical fluid chromatography (SFC) and to the nature of carbon dioxide, which is not polar, thus SFC is classified as a normal-phase separation technique. Moreover, additional effects are not well known and described. They are mainly related to density changes of the mobile phase or to adsorption of fluid on the stationary phase causing a modification of its surface. It is admitted that pressure or temperature modifications induce variation in the eluotropic strength of the mobile phase, but effects of flow rate or column length on retention factor changes are more surprising. Nevertheless, the retention behaviour in SFC first depends on the stationary phase nature. Working with polar stationary phases induces normal-phase retention behaviour, whereas using non-polar bonded phases induces reversed-phase retention behaviour. These rules are verified for most carbon dioxide-based mobile phases in common use (CO(2)/MeOH, CO(2)/acetonitrile or CO(2)/EtOH). Moreover, the absence of water in the mobile phase favours the interactions between the compounds and the stationary phase, compared to what occurs in hydro-organic liquids. Other stationary phases such as aromatic phases and polymers display intermediate behaviours. In this paper, all these behaviours are discussed, mainly by using log k-log k plots, which allow a simple comparison of stationary phase properties. Some examples are presented to illustrate these retention properties.     

分子印迹手性分离过程的热力学研究

以丙烯酰胺为功能单体, 以二甲基丙烯酸乙二醇酯为交联剂, 在模板分子N-叔丁氧羰酰-L-色氨酸(N-Boc-L-Trp)和N-叔丁氧羰酰-L-酪氨酸(N-Boc-L-Tyr)的存在下, 分别采用光引发聚合和热引发聚合制备了N-Boc-L-Trp和N-Boc-L-Tyr的分子印迹聚合物(MIPs), 进行分子印迹手性分离过程的热力学研究. 测定了分离过程的熵变、焓变和自由能变化. 结果显示, 在流动相中添加异丙醇或甲醇等强氢键竞争性溶剂时, 熵变对分离起到了主要作用, 而且分离过程中的溶剂化对分离的影响也非常大. 分子印迹聚合物对印迹分子和非印迹分子进行分子识别的主要作用是印迹聚合物与印迹分子匹配的三维空间结构.